Methods of treating intestinal ischemia using heparin-binding epidermal growth factor

ABSTRACT

The present invention provides methods of treating pathologic conditions associated with intestinal ischemia. In the methods, patients at risk for or suffering from intestinal ischemia are treated with a heparin-binding epidermal growth factor product.

[0001] This application claims the benefit of the filing date of U.S.provisional application Serial No. 60/063,858.

FIELD OF THE INVENTION

[0002] The present invention generally relates to prevention and/ortreatment of ischemia-induced intestinal injury. More particularly theinvention relates to prevention and/or treatment of intestinal injuryusing heparin-binding epidermal growth factor (HB-EGF) products.

BACKGROUND OF THE INVENTION

[0003] Hemorrhagic disorders and ischemic states are the two majorclasses of gastrointestinal circulatory disorders. A sudden reduction inthe blood supply to a tissue is considered to be an ischemic event.Intestinal ischemic events continue to play a major role in themorbidity and mortality of numerous patients. Ischemic injury to thesmall intestine results in mucosal destruction, bacterial translocation,and perforation. Parks et al., Am. J. Physiol., 250: G749-753 (1986)attributes much of the injury associated with ischemic episodes to thereperfusion phenomena that begin when blood flow is restored.Immediately after an ischemic event, the intestinal epithelium undergoesdesquamation with destruction of the lamina propria. At a cellularlevel, ischemia leads to depletion of ATP and loss of cytoskeletalintegrity. With return of the blood supply (i.e., reperfusion), there iscontinued destruction of the villus structures. These injuries manifestthemselves in disease states such as necrotizing enterocolitis and canlead to overwhelming sepsis and multisystem organ failure. Recovery froman ischemic event depends on rapid proliferation and migration ofintestinal epithelial cells to regenerate damaged villi. Restitutionrequires the presence of multiple substances, including cytokines,hormones, and growth factors. Dignass and Podolsky, Gastroenterology,105: 1323-1332 (1993) reports that transforming growth factor-α (TGF-α),interleukin-1β (IL-1β), interferon-γ (IFN-γ), and epidermal growthfactor (EGF) have been shown to enhance restitution, possibly throughincreased production of transforming growth factor-β (TGF-β). Thesesubstances act to remodel the intestine after injury and to modulate theinflammatory response.

[0004] HB-EGF was originally identified in 1990 as a macrophage-secretedheparin binding growth factor. Like other members of the EGF family,HB-EGF exerts its biological effects by binding to the erb class of EGFreceptor (EGF-R) molecules. However, unlike most members of the EGFfamily including EGF, HB-EGF binds heparin with a high affinity. Heparinappears to potentiate binding of HB-EGF to the signal-transducing EGF-R,and may also modulate the biologic effects of the growth factor ontarget cells, including cellular migration and proliferation. HB-EGF ismitogenic for fibroblasts, smooth muscle cells and epithelial cells, butnot for endothelial cells. In addition, HB-EGF is produced by epithelialcells and acts as an autocrine growth factor for these cells. It is aheat-resistant, cationic protein, with a molecular weight ofapproximately 22,000 kDa that elutes from heparin-affinitychromatography columns with 1.0 M NaCl.

[0005] The cloning of a cDNA encoding human HB-EGF (or HB-EHM) isdescribed in Higashiyama et al., Science, 251: 936-939 (1991) and in acorresponding international patent application published under thePatent Cooperation Treaty as International Publication No. WO 92/06705on Apr. 30, 1992. Both publications are hereby incorporated by referenceherein. The sequence of the protein coding portion of the cDNA is setout in SEQ ID NO: 1 herein, while the deduced amino acid sequence is setout in SEQ ID NO: 2. Mature HB-EGF is a secreted protein that isprocessed from a transmembrane precursor molecule (pro-HB-EGF) viaextracellular cleavage. The predicted amino acid sequence of the fulllength HB-EGF precursor represents a 208 amino acid protein. A span ofhydrophobic residues following the translation-initiating methionine isconsistent with a secretion signal sequence. Two threonine residues(Thr⁷⁵ and Thr⁸⁵ in the precursor protein) are sites forO-glycosylation. Mature HB-EGF consists of at least 86 amino acids(which span residues 63-148 of the precursor molecule), and severalmicroheterogeneous forms of HB-EGF, differing by truncations of 10, 11,14 and 19 amino acids at the N-terminus have been identified. HB-EGFcontains a C-terminal EGF-like domain (amino acid residues 30 to 86 ofthe mature protein) in which the six cysteine residues characteristic ofthe EGF family members are conserved and which is probably involved inreceptor binding. HB-EGF has an N-terminal extension (amino acidresidues 1 to 29 of the mature protein) containing a highly hydrophilicstretch of amino acids to which much of its ability to bind heparin isattributed. Besner et al., Growth Factors, 7: 289-296 (1992), which ishereby incorporated by reference herein, identifies residues 20 to 25and 36 to 41 of the mature HB-EGF protein as involved in binding cellsurface heparin sulfate and indicates that such binding mediatesinteraction of HB-EGF with the EGF receptor.

[0006] The EGF family comprises at least five polypeptides: EGF, HB-EGF,TGF-α, amphiregulin (AR), and betacellulin. For reviews of the family,see Barnard et al., Gastroenterology, 108: 564-580 (1995) and Prigentand Lemoine, Prog. Growth Factor Res., 4: 1-24 (1992). The amino acidsequence homology of HB-EGF to the EGF family members is 40 (compared toEGF) to 53% (compared to AR) between the first and sixth cysteineresidues in the EGF-like domains, but HB-EGF exhibits lower homologywhen the full length sequences are compared. Overall, HB-EGF mostclosely resembles AR in that the two polypeptides exhibit the highesthomology, appear to have a similar number of amino acids, and includethe N-terminal extension of highly hydrophilic amino acids upstream ofthe EGF-like domain.

[0007] Administration of EGF to prevent tissue damage after an ischemicevent in the brains of gerbils has been reported in U.S. Pat. No.5,057,494 issued Oct. 15, 1991 to Sheffield. The patent projects thatEGF “analogs” having greater than 50% homology to EGF may also be usefulin preventing tissue damage and that treatment of damage in myocardialtissue, renal tissue, spleen tissue, intestinal tissue, and lung tissuewith EGF or EGF analogs may be indicated. However, the patent includesno experimental data supporting such projections.

[0008] The small intestine receives the majority of its blood supplyfrom the SMA, but also has a rich collateral network such that onlyextensive perturbations of blood flow lead to pathologic states. Villaet al., Gastroenterology, 110(4 Suppl): A372 (1996) reports that in arat model of intestinal ischemia in which thirty minutes of ischemia arecaused by occlusion of the superior mesenteric artery (SMA),pre-treatment of the intestines with EGF attenuated the increase inintestinal permeability compared to that in untreated rats. Theintestinal permeability increase is an early event in intestinal tissuechanges during ischemia. Multiple animal models, like that described inVilla et al., supra have been used to study the effects of ischemicinjury to the small bowel. Since the small intestine has such a richvascular supply, researchers have used complete SMA occlusion to studyischemic injury of the bowel. Animals who experience total SMA occlusionsuffer from extreme fluid loss and uniformly die from hypovolenia andsepsis, making models of this type useless for evaluating the recoveryfrom intestinal ischemia. Nevertheless, the sequence of morphologic andphysiologic changes in the intestines resulting from ischemic injury hasremained an area of intense examination.

[0009] Miyazaki et al., Biochem Biophys Res Comm, 226: 542-546 (1996)discusses the increased expression in a rat gastric mucosal cell line ofHB-EGF and AR resulting from oxidative stress. The authors speculatethat the two growth factors may trigger the series of reparative eventsfollowing acute injury (apparently ulceration) of the gastrointestinaltract. To date, there has been no published report of administration ofHB-EGF in vivo for any purpose, much less to test its ability to protectthe gastrointestinal tract from injury from an ischemic event.

[0010] The prevention and treatment of ischemic damage in the clinicalsetting therefore continues to be a challenge in medicine. There thusexists a need in the art for models for testing the effects of potentialmodulators of ischemic events and for methods of preventing and/ortreating ischemic damage, particularly ischemic damage to theintestines.

SUMMARY OF THE INVENTION

[0011] In a first aspect, the invention provides methods of treatingpathological conditions associated with intestinal ischemia byadministering an HB-EGF product to patients.

[0012] As used herein, “HB-EGF product” includes HB-EGF proteinscomprising about amino acid 63 to about amino acid 148 of SEQ ID NO: 2;HB-EGF proteins comprising about amino acid 73 to about amino acid 148of SEQ ID NO: 2; HB-EGF proteins comprising about amino acid 74 to aboutamino acid 148 of SEQ ID NO: 2; HB-EGF proteins comprising about aminoacid 77 to about amino acid 148 of SEQ ID NO: 2; HB-EGF proteinscomprising about amino acid 82 to about amino acid 148 of SEQ ID NO: 2;HB-EGF proteins comprising a continuous series of amino acids of SEQ IDNO: 2 which exhibit less than 50% homology to EGF and which areefficacious in the rat model specified below; fusion proteins comprisingthe foregoing HB-EGF proteins; and the foregoing HB-EGF proteinsincluding conservative amino acid substitutions. Conservative amino acidsubstitions are understood by those skilled in the art. The HB-EGFproducts may be isolated from natural sources known in the art [e.g.,the U-937 cell line (ATCC CRL 1593)], chemically synthesized, orproduced by recombinant techniques such as disclosed in WO92/06705,supra, the disclosure of which is hereby incorporated by reference. Inorder to obtain HB-EGF products of the invention, HB-EGF precursorproteins may be proteolytically processed in situ. The HB-EGF productsmay be post-translationally modified depending on the cell chosen as asource for the products.

[0013] The administration of HB-EGF products is preferably accomplishedwith a pharmaceutical composition comprising an HB-EGF product and apharmaceutically acceptable carrier. The carrier may be in a widevariety of forms depending on the route of administration. The route ofadministration may be oral, rectal, parenteral, or through a nasogastrictube. Examples of parenteral routes of adminstration are intravenous,intraperitoneal, intramuscular, or subcutaneous injection. The presentlypreferred route of administration is the oral route as the presentinvention contemplates that the acid stability of HB-EGF is a uniquefactor as compared to, for example, EGF. The HB-EGF pharmaceuticalcomposition may also include other ingrediants to aid solubility, or forbuffering or preservation purposes. Pharmaceutical compositioncontaining HB-EGF products comprises HB-EGF at a concentration of about0.5 to 10 mg/ml and preferably at a concentration of 1 mg/ml in saline.Addition of other bioactive compounds [e.g., antibiotics, free radicalscavenging or conversion materials (e.g., vitamin E, beta-carotene, BHT,ascorbic acid, and superoxide dimutase), fibrolynic agents (e.g.,plasminogen activators), and slow-release polymers] to the HB-EGFcompounds or separate administration of the other bioactive compounds isalso contemplated.

[0014] As used herein, “pathological conditions associated withintestinal ischemia” includes conditions which directly or indirectlycause intestinal ischemia (e.g., premature birth; birth asphyxia;congenital heart disease; cardiac disease; polycythemia; hypoxia;exchange transfusions; low-flow states; atherosclerosis, embolisms orarterial spasms; ischemia resulting from vessel occlusions in othersegments of the bowel; ischemic colitis; and intestinal torsion such asoccurs in infants and particularly in animals) and conditions which aredirectly or indirectly caused by intestinal ischemia (e.g., necrotizingenterocolitis, shock, sepsis, and intestinal angina). Thus, the presentinvention contemplates administration of HB-EGF products to patients inneed of such treatment including patients at risk for intestinalischemia, patients suffering from intestinal ischemia, and patientsrecovering from intestinal ischemia. The administration of HB-EGF topatients is contemplated in both the pediatric and adult populations.

[0015] More particularly, the invention contemplates a method ofreducing necrosis associated with intestinal ischemia comprisingadministering an HB-EGF product to a patient at risk for, sufferingfrom, or recovering from intestinal ischemia. Also contemplated is amethod of protecting intestinal epithelial cells from hypoxia comprisingexposing the cells to an HB-EGF product. Administration of, or exposureto, HB-EGF products reduces lactate dehyrogenase efflux from intestinalepithelial cells, maintains F-actin structure in intestinal epithelialcells, increases ATP levels in intestinal epithelial cells, and inducesproliferation of intestinal epithelial cells.

[0016] In view of the efficacy of HB-EGF in protecting intestinal tissuefrom ischemic events, it is contemplated that HB-EGF has a similarprotective effect on myocardial, renal, spleen, lung, and liver tissue.

[0017] In another aspect, the invention provides a novel animal model ofintestinal ischemia, designated herein a model of “segmental” intestinalischemia, that is useful for evaluating the efficacy of putativetherapeutics. Mammals, preferably rats, are subjected to reversiblearterial occlusion, wherein a first order branch of the SMA and terminalcollateral branches are occluded. Preferably, the first order branch ofthe SMA is selected from the group consisting of the middle ileum andthe distal ilieum. Also preferably, six to seven terminal collateralbranches are occluded. Reversible occlusion may be accomplished by meanssuch as a micro-vascular clip or sutures.

DETAILED DESCRIPTION OF THE INVENTION

[0018] Practice of the methods of the present invention is illustratedin the following examples wherein Example 1 describes production ofHB-EGF by recombinant techniques and demonstrations of activity of therecombinant protein in various assays including cytoprotection ofintestinal epithelial cells from hypoxia in vitro; Example 2 discloses asegmental model of intestinal ischemia in the rat; Example 3 describesthe efficacy of HB-EGF in treating intestinal ischemia in the rat model;and Example 4 details treatment of human adults and infants withpathological conditions associated with intestinal ischemia with HB-EGF.

EXAMPLE 1

[0019] The effects of treatment of rat intestinal epithelial cells withrecombinant human HB-EGF were examined. Specifically, HB-EGF was testedfor the ability to induce proliferation of intestinal endothelial cellsand also for the ability to protect intestinal epithelial cells fromhypoxia. Experiments were also performed to examine the mechanism ofHB-EGF cytoprotection. The rat intestinal epithelial cells IEC-18 (ATCCCRL 1589) were used in the experiments.

[0020] A. Production of Recombinant Human HB-EGF (rHB-EGF)

[0021] The maltose-binding protein (MBP) fusion system (New EnglandBiolabs, Beverly, Mass.) was used to produce recombinant human HB-EGF.HB-EGF cDNA corresponding to nucleotides 220 to 444 of SEQ ID NO: 1(encoding amino acids 74-148 of the 208-amino acid HB-EGF precursormolecule) was cloned into plasmid pMAL-c2 at the Xmnl and HindIII sites.E. coli strain BL21 (F ompT r_(B) ⁻ m₈ ⁻) (Novagen, Madison, Wis.)containing this construct was grown at 37° C. in LB broth (Gibco/BRL,Gaithersburg, Md.) containing 2 g/L glucose (Gibco/BRL) and 100 μg/mlampicillin (Sigma, St. Louis, Mo.) to an OD₆₀₀ of 0.2-0.3. To induceexpression, IPTG (Promega, Madison, Wis.) was added to a finalconcentration of 0.3 mM. After a 3 hour incubation at 37° C., cells wereharvested by centrifugation (4,000×g, 20 minutes) and the cell pelletwas resuspended in MBP buffer (10 mM Tris-Ci, 200 mM NaCl, 1 mM EDTA)containing 1 mM PMSF (Sigma) and frozen overnight at 20° C. Thawed cellsample was lysed with a french press (14,000 psi) and the insolublefraction was removed by centrifugation (9,000×g, 30 minutes). Thesupernatant was passed over an amylose resin column (New EnglandBiolabs) and fusion protein was eluted in MBP buffer containing 10 mMmaltose (Sigma). rHB-EGF was cleaved from MBP with Factor Xa (0.5%, w/w)(Boehringer Mannheim, Indianapolis, Ind.) at 23° C. for 16 hours.Cleaved products were applied to a TSK-heparin 5PW column (8×75 mm,TosoHaas, Philadelphia, Pa.) that was equilibrated with buffer (10 mMTris-HCI pH 7.4, 0.2M NaCl). The column was washed with equilibrationbuffer and bound proteins were eluted with a 40 ml linear gradient of0.2-2.0M NaCl in 10 mM Tris-HCI pH 7.4 at 1 ml/min using an FPLC system(Pharmacia LKB Biotechnology, Piscataway, N.J.). One milliliterfractions are collected and assayed in an EGF radioreceptor assayessentially as described in Besner et al. (1992), supra. Fractionsshowing peak displacement of ¹²⁵I-EGF binding were pooled, adjusted tocontain 5% acetonitrile and 0.1% trifluoroacetic acid and subjected toreverse phase HPLC (RP-HPLC). RP-HPLC was performed in a Hitachi (SanHose, Calif.) HPLC system using a Vydac C₄ column (0.46×25 cm, 5 μmparticle size; The Separations Group, Hesperia, Calif.) that wasequilibrated with water containing 5% acetonitrile and 0.1%trifluoroacetic acid. The HB-EGF sample was injected onto the column andthe column was eluted using a multilinear gradient of 5% acetonitrile(isocratic) for 5 minutes, 5-15% acetonitrile over 5 minutes, 15-40%over 120 minutes, 40-90% over 1 minute, 90% isocratic for 10 minutes,90-5% over 1 minute, and 5% isocratic for 33 minutes. The flow rate was1 ml/min throughout and 1 ml fractions were collected. After thesepurification steps, the absorbance peak eluting at 19% acetonitrile isshown by SDS-PAGE to be a single band migrating at approximately 13 kDA.NH₂-terminal amino acid sequencing of the pure protein produced in thisfashion confirms the sequence for HB-EGF. The rHB-EGF was biologicallyactive in the EGF-radioreceptor assay and a Balb/c 3T3 DNA synthesisassay [essentially as described in Besner et al., Cell Regulation, 1:811-819 (1990)].

[0022] B. Recombinant HB-EGF is Mitogenic for Intestinal EpithelialCells

[0023] IEC-18 cells were cultured in Dulbecco's Modified Eagle Medium(DMEM) containing 5% fetal bovine serum (FBS), 50 μg/ml penicillin and50 units/ml streptomycin in a humidified atmosphere of 10% CO₂ at 37° C.Cells were then seeded at a density of 1×10⁵ cells/well in a 24 wellplate (2 ml/well). After a 24 hour incubation, medium was changed toDMEM/1% FBS (2 ml/well) and cells were allowed to incubate for anadditional 24 hours. Cells in four of the wells were trypsinized andcounted at Day 0 using a Coulter Counter®. rHB-EGF was added toduplicate wells on Day 0 at a concentration of 100 ng/ml. Thisconcentration of rHB-EGF was determined as optimal from preliminarydose-response curves for rHB-EGF-stimulated proliferation of IEC-18cells. Cells in duplicate wells were trypsinized and counted on days 1through 5.

[0024] Under normoxic conditions, rHB-EGF-treated cells had 1.85 foldgreater increase in number compared to non-treated cells by day 4(p<0.05). Further, the mitogenic response of IEC-18 cells to rHB-EGF wasdose-dependent, with maximal stimulation by 100 ng/ml.

[0025] C. Recombinant HB-EGF to Protects Intestinal Epithelial CellsAgainst Hypoxia

[0026] Lactate dehyrogenase (LDH) efflux was used as a measure of cellinjury after hypoxia. IEC-18 cells were seeded at a density of 5×10⁴cells/well in DMEM/5% FBS in 24 well plates (2 ml/well). After 24 hours,medium was changed to DMEM/1% FBS (2 ml/well). This plating densityresulted in approximately 75% confluency. After an additional 24 hourincubation period, the medium was replaced with Kreb's buffer (116 mMNaCl, 1.0 mM NaH₂PO₄, 25.0 mM NaHCO₃, 5.4 mM KCl, 1.8 mM CaCl₂, and 0.8mM MgSO₄) and the cells were placed in an anaerobic incubator withFiO₂≦1%. Aliquots (50 μl) of media were removed at specific timeintervals and were assayed for LDH efflux using a Cytox 96 assay. At theend of the experiment, cells were lysed with PBS/0.1% Triton-100 andtotal LDH content was determined. LDH efflux was expressed as thepercentage of total LDH activity. Based on preliminary experiments whichdemonstrated approximately 20-25% cell death after 10 hours of hypoxiaand 100% cell death after 14 hours of hypoxia, a 10 hour anaerobicperiod was used in the subsequent studies.

[0027] To test the growth factor effects, 100 ng/ml rHB/EGF was added tosome wells 12 hours prior to the initiation of hypoxia. After 10 hoursof hypoxia, plates were removed from the anaerobic chamber, mediumchanged to DMEM/5% FBS, and cells were allowed to recover for 48 hours.During recovery, some wells received additional (post-hypoxia) rHB-EGF(100 ng/ml) treatment. Aliquots (50 μl) of media were removed from thewells at 0, 12, 24, 36, and 48 hours of recovery and LDH efflux wasmeasured.

[0028] Intestinal cells that received rHB-EGF either pre-hypoxia or bothpre- and post-hypoxia showed a significantly lower LDH release duringrecovery from hypoxia compared to non-treated cells. Although there wasvery little LDH efflux immediately after hypoxia, by 48 hours,non-treated cells had an LDH release of 22.8% compared to 7.48% forcells that had been pre-treated with HB-EGF (p<0.009) or to 9.1% forcells that had been both pre-treated and post-treated with HB-EGF(P<0.009). Cells that received HB-EGF during only the post-hypoxicperiod did not have a significantly lower LDH release compared to cellsthat were not treated with HB-EGF.

[0029] D. Effects of rHB-EGF on Cytoskeletal Structure, ATP Stores, andPost-Hypoxia Proliferation

[0030] To examine IEC-18 cytoskeletal structure, IEC-18 cells wereseeded at 5×10³ cells/well in DMEM/5% FBS (500 μl/chamber) in 8-wellchamber slides. Cells were incubated for 24 hours after which the mediumwas changed to DMEM/1% FBS. rHB-EGF (100 ng/ml) was then added tospecific wells. After an additional 12 hours, medium was changed toKreb's buffer and cells were placed in the anaerobic chamber for 10hours. Following this, medium was changed to DMEM/5% FBS and cells wereincubated for an additional 24 or 48 hours. Medium was aspirated fromthe chambers, cells were fixed for thirty minutes in 10% formalin(buffered with PBS), and slides were washed twice in PBS. Cells weresimultaneously stained with rhodamine phalloidin to detect filamentous(F) actin and Dnase I fluorescein to detect globular (G) actin. Confocalanalysis of the cells was performed with the Zeiss LSM invertedmicroscope using a krypton/argon mixed gas laser and a Zeiss filter set,utilizing emissions filters of 568 and 488. Images were digitallyrecorded.

[0031] Under normal circumstances, monomeric G-actin is polymerized toproduce F-actin in an ATP-dependent manner. F-actin staining of cells ispresent in the cortical region (peripheral) in cells having an intactcytoskeleton, whereas G-actin accumulation is indicative of cytoskeletalinjury. Immediately after anaerobic exposure, the respective proportionsof F-actin and G-actin were similar between rHB-EGF-treated andnon-treated cells. However, after 24 hours of recovery, IEC-18 cellsthat received rHB-EGF prior to anaerobic exposure maintained thecortical F-actin cytoskeletal structure compared to non-treated cellswhich had increase levels of peri-nuclear G-actin staining. By 48 hours,rHB-EGF-treated cells still maintained their F-actin structure, whereasnon-treated cells contained predominately G-actin with very littleF-actin.

[0032] To examine ATP stores in IEC-18 cells, the cells were seeded at adensity of 1×10⁵ cells/well in DMEM/5% FBS in 6 well palates (1ml/well). Cells were incubated for 24 hours after which the medium waschanged to DMEM/1% FBS. rHB-EGF (100 ng/ml) was then added to specificwells. After an additional 12 hours, medium was changed to Kreb's bufferand cells were placed in the anaerobic chamber for 10 hours. Cells werelysed with PBS/0.1% Triton-100 at 0, 24 and 48 hours of recovery. ATPcontent was measured using an ATP determination kit (Molecular Probes,Eugene, Oreg.). This assay allows quantification of ATP through aluciferin/luciferase-ATP reaction. Bioluminescence was measured with aluminometer (Model LB9501). Initial experiments demonstrated a linearrelationship (r²=0.960) between ATP (pmoles) and luminescence. A Bio-RadD_(c) protein assay was used to determine total protein content in thesamples. Optical density (OD) was measured with a microplate reader(Model EL312). Preliminary standard curves demonstrated a linearrelationship (r²=0.988) between protein (mg/ml) and OD.

[0033] Both non-treated and rHB-EGF treated cells had ATP levels in the11-12 nmole/mg range under normoxic conditions. rHB-EGF-treated andnon-treated cells had similar decreases in ATP levels in the immediatepost-hypoxic period (45% drop vs 50% drop, respectively). However,during the later recovery periods (24 and 48 hours), the HB/EGF-treatedcells exhibited a rise in their ATP levels (6.1 nmole/mg), whereas thenon-treated cells continued to have decreased ATP content (4.5nmole/mg).

[0034] Post-hypoxia IEC-18 cell proliferation was assessed with aCytoQuant fluorescence assay (Molecular Probes, Eugene, Oreg.).Preliminary experiments demonstrated a linear relationship (r²=0.978)between cell number and fluorescence. IEC-18 cells were seeded at 5×10³cells/well in DMEM/5% FBS in 96-well plates (200 μl/well). After 24hours of incubation, medium was changed to DMEM/1% FBS (200 μl/well).Some wells received HB-EGF (100 ng/ml) and the cells were incubated foran additional 12 hours. Medium was then changed to Kreb's buffer and theplate was placed in the anaerobic chamber for 10 hours. At 0, 12, 36,and 72 hours of recovery, media was removed and plates were frozen at−70° C. for thirty minutes. The Cytoquant fluoroprobe was added afterincubating plate at room temperature for five minutes. Fluorescence wasmeasured in a Cytofluor fluorescent plate reader (Millipore, Bedford,Mass.).

[0035] Cytofluorometric measurement of post-hypoxia cellularproliferation of IEC-18 cells showed that cells treated with HB-EGF hada 1.23 fold increase in a cell number compared to non-treated cells by72 hours of recovery from hypoxia (p<0.05).

[0036] The experimental results described above relating to themitogenic and cytoprotective effects of HB-EGF were orally disclosed atthe Columbus Surgical Society Presidential Symposium on Jan. 18, 1997and at the Annual West Virginia University Resident's Forum on Mar. 7,1997. The experimental results described above relating to LDH releasewere disclosed orally and in abstract form at the Childrens' HospitalResearch Foundation Research Forum in Columbus, Ohio on Jun. 5, 1997.

[0037] HB-EGF was also demonstrated to preserve cytoskeletal structureand increase cellular ATP levels in renal tubular epithelial cellssubject to hypoxia. The same cells also released a lower level of LDHduring recovery than untreated cells.

[0038] The results of the in vitro experiments indicate that in additionto being a mitogen for intestinal epithelial cells, HB-EGF is also acytoprotective growth factor for these cells during recovery fromhypoxia. The in vitro cytoprotective effects of rHB-EGF can beexplained, at least in part, by increased cellular ATP levels inrHB-EGF-treated cells with resultant preservation of cytoskeletalstructure. An additional beneficial effect of this growth factor is thatafter the ischemic event, HB-EGF-treated cells have a higherproliferative rate than non-treated cells. The cytoprotective effects ofHB-EGF may be enhanced by its ability to bind to heparan sulfateproteoglycans expressed on the surface of intestinal cells. See Carey etal., J. Cell Biology, 117(1): 191-201 (1992) for a discussion of heparansulfate proteoglycans.

EXAMPLE 2

[0039] While HB-EGF protected intestinal epithelial cells from hypoxiain vitro, there was no model described in the literature that was usefulfor examining the effect of HB-EGF on recovery from intestinal ischemiain vivo. To determine whether HB-EGF was efficacious in protecting theintestines from the deleterious effects of ischemia in vivo, a novelanimal model of segmental intestinal ischemia was developed. The modelprovides the opportunity to study ischemia-reperfusion injuries inlocalized segments of bowel without the morbidity and mortalityassociated with total SMA occlusion in prior animal models. By occludinga first order branch of the superior mesenteric artery (SMA) and byselectively ligating terminal collateral branches, reproduciblesegmental intestinal ischemia was achieved. Bowel damage ranged fromalterations in the villus structure to frank hemorrhagic necrosis of theintestinal wall.

[0040] The operative procedure was performed as follows. A total ofeighteen rats (age 7 to 9 weeks, 200-265 g) were induced with anintraperitoneal injection of Ketamine-HCl (10 mg/kg) and Xylazine (3mg/kg). Intravenous access and/or fluid resuscitation were not necessaryduring the procedure. The abdomen was shaved and painted with betadine.The animal was placed supine on a warming pad set at 40° C., andpositioned under an operating microscope. A midline skin incision wasmade, the linea alba was opened, and the peritoneal cavity was entered.The small intestine, cecum and proximal ascending colon were deliveredinto the operative field and the superior mesenteric vein wasidentified. The SMA was located postero-lateral to the vein and wasexposed by carefully dissecting apart the mesentery with 0.5 mmmicro-surgical forceps. Exposure of this artery and the subsequent stepswere performed using an operating microscope. Once the SMA was exposed,the first order branches were evaluated for possible sites of occlusion.Since the mesenteric arcades are longer in the ileal segments, themiddle and distal ileum were used as target segments for arterialocclusion. Once a segment was identified, a micro-vascular atraumaticclip (2.0 mm) was placed on the first order mesenteric branch feedingthis segment. After the clip was placed, the terminal arterial andvenous arcade branches, both proximal and distal to the occluded arcade,were ligated with 5.0 silk sutures. To create a 5 cm segment of ischemicbowel, six to seven terminal branches need to be ligated. Arterialocclusion was maintained for 1 hour. A 4×4 gauze pad was placed over thebowel during this time and was frequently moistened with warm saline.Prior to removing the micro-clip, 0.1% Evan's blue solution (1.5 cc) wasinjected into the renal vein with a 28 gauge needle to confirmnon-perfusion of the ischemic segment. The micro-clip was then removedand Evan's blue, at the same concentration, was injected into thecontralateral renal vein to establish return of flow to the ischemicsegment. The silk ligatures were left on the terminal branches. Theabdomen was closed in a standard fashion. Animals were then placed in awarm incubator (40° C.) until awake (approximately 20 minutes) and werethen transferred to individual cages. They received water, but not food,during the post-operative period. Animals were euthanized with CO₂ andsegments of intestine were removed for histologic analysis at 6 hoursafter surgery in 6 animals and at 48 hours after surgery in 12 animals.

[0041] All eighteen animals survived the operation. Gross changes of thebowel during the arterial occlusion were noted to occur in stages. After10-15 minutes of ischemia the serosa loses its sheen, and after 15-20minutes the bowel wall becomes edematous. After 25-35 minutes the bowelcolor changes from pink to white and later develops a more duskyappearance. The proximal and distal ends of the segment, where theterminal arteries and veins were ligated, have a more bluish appearance,and the smaller veins become dilated. Peristalsis of the affectedsegment ceases within the first 25 minutes of ischemia. As ischemic timeincreases, the bowel wall becomes increasingly edematous. Confirmationof ischemia was shown with Evan's blue dye injection, with the dye takenup by the normally perfused bowel, but not by the ischemic segment. Upontermination of arterial occlusion, uptake of the blue dye throughout thepreviously ischemic bowel confirmed resumption of flow to this area.

[0042] After euthanization, the abdomen was re-explored and the segmentof ischemic bowel, as well as portions of intestine both proximal anddistal to the hypo-perfused segment, were excised and fixed inHistochoice™ for 12 hours. The segments were then cross-sectioned at 1mm intervals, processed in a standard fashion, and embedded in paraffin.Sections were H&E stained and examined using a standard lightmicroscope. All six animals that were sacrificed at the 6 hour timepoint showed minor intestinal villus structural change. Instead of thenormal elongated distal tip, the villi had more flattened tips withreduction of cytoplasmic content and absence of the brush border. Oftwelve animals sacrificed after 48 hours, all showed evidence of villustip necrosis, and five also developed areas of hemorrhagic transmuralnecrosis with extensive polymorphonuclear leukocyte infiltration.

[0043] Animal care and experimentation described above conformed tostandards listed in the National Institute of Health's Guide for theCare and Use of Laboratory Animals. The experimental protocol wasevaluated and approved by the Institutional Animal Care and UseCommittee of Children's Hospital (protocol #01496AR). Each procedure wasperformed by a single operator without the need for additionalassistance. All procedures were performed using sterile technique. Theduration of the procedure was 1¼-1½ hours, depending on the difficultyof the dissection.

[0044] In comparison to ischemia-reperfusion injury models involvingtotal SMA occlusion the mortality rate of which approaches 100%, thesegmental model described herein had a 0% postoperative mortality in thefirst 48 hours. The development of the segmental model thus allowed theinvestigation of the effects of potential therapeutic agents during therecovery period.

EXAMPLE 3

[0045] The effect of HB-EGF on intestinal ischemia in vivo was thenexamined using the segmental model.

[0046] A total of twelve rHB-EGF-treated and twelve control maleSprague-Dawley rats (220-310 g) were studied. Segmental intestinalischemia involving a 4.5-5.0 cm length of distal ileum was produced asdescribed in Example 2. Arterial occlusion was maintained for 1 hour.Fifteen minutes prior to removing the microvascular clip, Evan's bluesolution was injected intravenously to confirm segmental intestinalischemia. Experimental animals then received HB-EGP (20 μg/ml) which wasinjected intraluminally into the duodenum in a volume of 5 ml ofphosphate buffered saline (PBS), which led to filing of the entiregastrointestinal tract from the duodenum to the cecum. Control animalsreceived an injection of 5 ml of PBS only. At the end of the 1 hourischemic interval, the microvascular clamp was removed and the Evan'sblue solution was reinjected intravenously to confirm segmentalintestinal reperfusion. Animals received water but no foodpostoperatively. After 48 hours animals were sacrificed and necropsyperformed. The ischemic intestinal segment as well as the bowel justproximal and distal to the ischemic segment were excised. Tissues wereplaced in Histochoice fixative for 12 hours, cross-sectioned at randomintervals and embedded in paraffin. Sections were H & E stained andexamined using a standard light microscope. For each animal in theexperiment, 4-10 random sections of the ischemic bowel segment wereexamined for histologic injury. Results are presented in Table 1 below.TABLE 1 # sections Transmural Total # animals examined Tip necrosisnecrosis injury Non- 12 92 51/92 (55%) 21/92 (23%) 72/92 treated (78%)rHB- 12 94 12/94 (13%)  0/94 (0%)  12/94 EGF- (13%) treated

[0047] Of the non-treated animals, 55% of the representative sectionsstudied had villous tip necrosis, and 23% of the sections had transmuralnecrosis. In contrast, in the HB-EGF-treated animals, only 13% of thesections studied had villous tip necrosis, and none of the sectionsdisplayed transmural necrosis. Thus, intraluminal HB-EGF administration,in this case delivered 45 minutes after the initiation of the ischemicevent, protects the intestine from ischemic injury.

EXAMPLE 4

[0048] The following section exemplifies administration of HB-EGF topediatric patients and adult patients. Administration of HB-EGF isindicated in patients at risk for or suffering from any pathologicalcondition associated with ischemic injury.

[0049] A. Administration to Pediatric Patients

[0050] Intestinal injury related to an ischemic event is a major riskfactor for neonatal development of necrotizing enterocolitis (NEC). NECaccounts for approximately 15% of all deaths occurring after one week oflife in small premature infants. Although most babies who develop NECare born prematurely, approximately 10% of babies with NEC are full-terminfants. Babies with NEC often suffer severe consequences of the diseaseranging from loss of a portion of the intestinal tract to the entireintestinal tract. At present, there are no known therapies to decreasethe incidence of NEC in neonates.

[0051] Babies considered to be at risk for NEC are those who arepremature (less than 36 weeks gestation) or those who are full-term butexhibit, e.g., prenatal asphyxia, shock, sepsis, or congenital heartdisease. The presence and severity of NEC is graded using the stagingsystem of Bell et al., J. Ped. Surg., 15:569 (1980) as follows: Stage IAny one or more historical factors producing perinatal stress (SuspectedSystemic manifestations - temperature instability, lethargy, NEC) apnea,bradycardia Gastrointestinal manifestations - poor feeding, increasedpregavage residuals, emesis (may be bilious or test positive for occultblood), mild abdominal distention, occult blood in stool (no fissure)Stage II Any one or more historical factors (Definite Above signs andsymptoms plus persistant occult or gross NEC) gastrointestinal bleeding,marked abdominal distention Abdominal radiographs showing significantintestinal disten- tion with ileus, small-bowel separation (edema inbowel wall or peritoneal fluid), unchanging or persistent “rigid” bowelloops, pneumatosis intestinalls, portal venous gas Stage III Any one ormore historical factors (Advanced Above sings and symptoms plusdeterioration of vital signs, NEC) evidence of septic shock, or markedgastrointestinal hemor- rhage Abdominal radiographs showingpneumoperitoneum in addi- tion to findings listed for Stage II

[0052] Babies at risk for or exhibiting HB-EGF are treated as follows.Patients receive a daily liquid suspension of HB-EGF (1 mg/kg insaline). The medications are delivered via a nasogastric tube if one isin place, or orally if there is no nasogastric tube in place.

[0053] B. Administration to Adult Patients

[0054] Adults also receive a daily liquid suspension containing HB-EGF(1 mg/kg) to drink or through a nasogastric tube if necessary.

[0055] Numerous modifications and variations in the practice of theinvention are expected to occur to those skilled in the art uponconsideration of the foregoing description. Consequently, the onlylimitations which should be placed on the invention are those whichappear in the following claims.

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We claim:
 1. A method of treating pathological conditions associatedwith intestinal ischemia comprising administering an HB-EGF product topatients in an amount effective to reduce intestinal cell necrosis.